S. aureus infections are treated with antibiotics, with penicillin being the drug of choice whereas vancomycin is used for methicillin resistant isolates. The percentage of staphylococcal strains exhibiting wide-spectrum resistance to antibiotics has become increasingly prevalent since the 1980's (Panlilo et al 1992, Infect. Control. Hosp. Epidemiol. 13; 582), posing a threat for effective antimicrobial therapy. In addition, the recent emergence of vancomycin resistant S. aureus strain has aroused fear that methicillin resistant S. aureus strains will emerge and spread for which no effective therapy is available.
An alternative approach of using antibodies against staphylococcal antigens in passive immunotherapy has been investigated. Therapy involving administration of polyclonal antisera are under development (WO 00/15238, WO 00/12132).
An alternative approach would be use of active vaccination to generate an immune response against staphylococci. Several candidates for inclusion as vaccine components have been identified. These include Fibronectin binding protein (U.S. Pat. No. 5,840,846), MHC II analogue (U.S. Pat. No. 5,648,240), fibrinogen binding proteins ClfA and ClfB (U.S. Pat. No. 6,008,341, WO 99/27109), GehD (US 2002/0169288), collagen binding protein (U.S. Pat. No. 6,288,214), SdrC, SdrD, SdrE, SdrF, SdrG and SdrH (WO 99/27109, WO 00/12689, WO 08/19162), mutant SEA and SEB exotoxins (WO 00/02523), 52 kDa vitronectin binding protein (WO 01/60852), IsdA, IsdB, IsdC and IsdH (WO 05/09379, WO 08/152,447).
Clumping factor A (ClfA) has been identified as a S. aureus fibrinogen binding protein (U.S. Pat. No. 6,008,341) and has been identified as a potential carrier protein for polysaccharides which cold be used to immunize against staphylococcal infection (WO 04/80490).
Recently, amino acids P336 and Y338 of ClfA have been recognised as fibrinogen binding sites, mutation of which led to the loss of fibrinogen binding (Josefsson et al 2008, PLOS One volume 3, Issue 5, page 1-7). The loss of fibrinogen binding in these variants led to an increased ability to protect against septic death in immunised mice, leading to the conclusion that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.
There remains a need to develop a vaccine which protects against staphylococcal disease. An approach using S. aureus capsular polysaccharide conjugates has failed to achieve regulatory approval (WO 03/61558) and a more complex vaccine containing additional staphylococcal components may be required to give effective protection.
Accordingly, there is provided a ClfA polypeptide, fragment thereof or fusion protein thereof wherein amino acid Y474 or an amino acid adjacent to Y474 is mutated such that fibrinogen binding activity is decreased compared to an equivalent ClfA polypeptide or fragment thereof or fusion protein thereof without mutation of Y474 or an amino acid adjacent to Y474 (i.e. the relevant wild type ClfA).
In a second aspect of the invention, there is provided a polynucleotide encoding a ClfA polypeptide or fragment or fusion protein thereof, wherein amino acid Y474 or an amino acid adjacent to Y474 is mutated such that fibrinogen binding activity is decreased compared to an equivalent ClfA polypeptide or fragment thereof or fusion protein thereof without mutation of Y474 or an amino acid adjacent to Y474 (i.e. the relevant wild type ClfA).
In a third aspect of the invention, there is provided an immunogenic composition comprising a ClfA polypeptide wherein amino acid Y474 or an amino acid adjacent to Y474 is mutated such that fibrinogen binding activity is decreased compared to an equivalent ClfA polypeptide or fragment thereof or fusion protein thereof without mutation of Y474 or an amino acid adjacent to Y474 (i.e. the relevant wild type ClfA); and a pharmaceutically acceptable excipient.
In a fourth aspect of the invention there is provided a process for making the immunogenic composition of the invention comprising a step of adding a pharmaceutically acceptable excipient to the ClfA polypeptide, fragment or fusion protein wherein amino acid Y474 or an amino acid adjacent to Y474 is mutated such that fibrinogen binding activity is decreased compared to a an equivalent ClfA polypeptide or fragment thereof or fusion protein thereof without mutation of Y474 or an amino acid adjacent to Y474 (i.e. the relevant wild type ClfA).
In a fifth aspect of the invention there is provided a ClfA polypeptide or fragment of fusion protein, wherein amino acid Y474 or an amino acid adjacent to Y474 is mutated such that fibrinogen binding activity is decreased compared to a an equivalent ClfA polypeptide or fragment thereof or fusion protein thereof without mutation of Y474 or an amino acid adjacent to Y474 (i.e. the relevant wild type ClfA) for use in the treatment or prevention of staphylococcal infection or disease.
In a sixth aspect of the invention, there is provided a use of a ClfA polypeptide or fragment or fusion protein wherein amino acid Y474 or an amino acid adjacent to Y474 is mutated such that fibrinogen binding activity is decreased compared to an equivalent ClfA polypeptide or fragment thereof or fusion protein thereof without mutation of Y474 or an amino acid adjacent to Y474 (i.e. the relevant wild type ClfA) in the preparation of a medicament for the treatment or prevention of staphylococcal disease.
In a seventh aspect of the invention, there is provided a method of treating or preventing staphylococcal disease comprising administering a ClfA polypeptide or fragment or fusion protein polypeptide wherein amino acid Y474 or an amino acid adjacent to Y474 is mutated such that fibrinogen binding activity is decreased compared to an equivalent ClfA polypeptide or fragment thereof or fusion protein thereof without mutation of Y474 or an amino acid adjacent to Y474 (i.e. the relevant wild type ClfA) to a patient in need thereof.